Simultaneous analysis of multiple urinary biomarkers for the evaluation of oxidative stress by automated online in-tube solid-phase microextraction coupled with negative/positive ion-switching mode liquid chromatography-tandem mass spectrometry.

This study described an automated online method for the simultaneous determination of 8-isoprostane, 8-hydroxy-2'-deoxyguanosine, and 3-nitro-l-tyrosine in human urine. The method involves in-tube solid-phase microextraction using a Carboxen 1006 PLOT capillary column as an extraction device, followed by liquid chromatography with tandem mass spectrometry using a CX column and detection in the negative/positive switching ion-mode by multiple reaction monitoring. Using their stable isotope-labeled internal standards, each of these oxidative stress biomarkers showed good linearity from 0.02 to 2.0 ng/mL. Their detection limits (S/N = 3) were 3.4-21.5 pg/mL, and their intra- and inter-day precisions (relative standard deviations) were >3.9 and 6.5% (n = 5), respectively. This method was applied successfully to the analysis of urine samples, without any other pretreatment and interference peaks.

Oxidation of 1-N2-etheno-2'-deoxyguanosine by singlet molecular oxygen results in 2'-deoxyguanosine: a pathway to remove exocyclic DNA damage?

Exocyclic DNA adducts are considered as potential tools for the study of oxidative stress-related diseases, but an important aspect is their chemical reactivity towards oxidant species. We report here the oxidation of 1-N2-etheno-2'-deoxyguanosine (1,N2-εdGuo) by singlet molecular oxygen (1O2) generated by a non-ionic water-soluble endoperoxide [N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide endoperoxide (DHPNO2)] and its corresponding oxygen isotopically labeled [18O]-[N,N'-di(2,3-dihydroxypropyl)-1,4- naphthalenedipropanamide endoperoxide (DHPN18O2)], and by photosensitization with two different photosensitizers [methylene blue (MB) and Rose Bengal (RB)]. Products detection and characterization were achieved using high performance liquid chromatography (HPLC) coupled to ultraviolet and electrospray ionization (ESI) tandem mass spectrometry, and nuclear magnetic resonance (NMR) analyses. We found that dGuo is regenerated via reaction of 1O2 with the ε-linkage, and we propose a dioxetane as an intermediate, which cleaves and loses the aldehyde groups as formate residues, or alternatively, it generates a 1,2-ethanediol adduct. We also report herein the quenching rate constants of 1O2 by 1,N2-εdGuo and other etheno modified nucleosides. The rate constant (kt) values obtained for etheno nucleosides are comparable to the kt of dGuo. From these results, we suggest a possible role of 1O2 in the cleanup of etheno adducts by regenerating the normal base.

Paper-based solid-phase microextraction for analysis of 8-hydroxy-2'-deoxyguanosine in urine sample by CE-LIF.

An easy-to-do paper-based solid-phase microextraction (p-SPME) was developed for determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine sample by CE-LIF. Small piece of filter paper was used as a solid phase to extract 8-OHdG from urine sample. Its primary mechanism is based on the hydrogen-bonding interaction between 8-OHdG and cellulose molecules. The effects of the pH of the sample solution, extraction time, and temperature on the peak area of the analyte were investigated in order to obtain the optimal p-SPME conditions. Comparing with the untreated sample, the p-SPME can significantly reduce the interference to the separation of 8-OHdG by CE-LIF. Meanwhile, the p-SPEM can provide more than three times concentrated effect. The developed method was evaluated according to an FDA guideline for biological analysis. The precisions (RSD%, n = 5) of the peak area and migration time of the analyte at three different concentrations were within 3.02-5.82% and 0.92-1.58%, respectively. The limit of identification of the method is about 5 nM according to the significant difference between two sets of the samples with and without spiking the standard (Student's t-test, p < 0.05). Good linearity was obtained in the range of 10-1000 nM (R2 >0.99) based on the standard addition. The recoveries at three different concentrations were within 99.8-103.5%. The results of the real sample analysis are consistent with those reported in our previous paper (Electrophoresis 2014, 35, 1873-1879).

Validation and development of MTH1 inhibitors for treatment of cancer.

BACKGROUND: Previously, we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidised nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently, several new and potent inhibitors of MTH1 were demonstrated to be non-toxic to cancer cells, challenging the utility of MTH1 inhibition as a target for cancer treatment. MATERIAL AND METHODS: Human cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA or shRNA. 8-oxodG was measured by immunostaining and modified comet assay. Thermal Proteome profiling, proteomics, cellular thermal shift assays, kinase and CEREP panel were used for target engagement, mode of action and selectivity investigations of MTH1 inhibitors. Effect of MTH1 inhibition on tumour growth was explored in BRAF V600E-mutated malignant melanoma patient derived xenograft and human colon cancer SW480 and HCT116 xenograft models. RESULTS: Here, we demonstrate that recently described MTH1 inhibitors, which fail to kill cancer cells, also fail to introduce the toxic oxidized nucleotides into DNA. We also describe a new MTH1 inhibitor TH1579, (Karonudib), an analogue of TH588, which is a potent, selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti-cancer properties in vivo. CONCLUSION: We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore, we describe TH1579 as a best-in-class MTH1 inhibitor, which we expect to be useful in order to further validate the MTH1 inhibitor concept.

Synthesis of 8-oxo-7,8-dihydro-2'-deoxyguanosine from 2'-deoxyguanosine using Cu(II)/H2O2/ascorbate: A new strategy for an improved yield.

The free nucleoside 2'-deoxyguanosine (dGuo) is the most susceptible to oxidation by reactive oxygen species (ROS) compared to the other free nucleosides, and its oxidation product 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) has been used as a biomarker to quantify oxidative stress damage. We investigated different reactions using Fe(2+) or Cu(2+) and H2O2 in order to identify the reaction with the best yield. HPLC coupled with a UV detector and micrOTOF mass spectrometry were used to detect and confirm the identity of 8-oxodGuo. The optimized reaction synthesized 8-oxodGuo with a yield of 72.0%, much higher than that previously described in the literature. Our improved method for 8-oxodGuo synthesis could be extremely useful for assays that require the synthesis of internal standards labeled with stable isotopes.

Automated Cell-Based Quantitation of 8-OHdG Damage.

Detection of 8-OHdG-base damage has been a big challenge for decades, though different analytical methods are developed. The recent approaches that are used for quantitating either the total amount of base damage or the amount of base damage per cell from different sources of samples are not automated. We have developed a method for automated damage detection from a single cell and applied it to 8-OHdG quantitation.

Value of the 8-oxodG/dG ratio in chronic liver inflammation of patients with hepatocellular carcinoma.

The aim of this study was to examine the role of oxidative DNA damage in chronic liver inflammation in the evolution of hepatocellular carcinoma. The accumulated data demonstrated that oxidative DNA damage and chronic liver inflammation are involved in the transformation of normal hepatocytes and their evolution towards hepatocellular carcinoma. However, the levels of 8-oxy-2'-deoxy-guanosine (8-oxodG), a biomarker of oxidative DNA damage, were overestimated and underestimated in previous reports due to various technical limitations. The current techniques are not suitable to analyze the 8-oxodG levels in the non-malignant liver tissues and tumors of hepatocellular carcinoma patients unless they are modified. Therefore, in this study, the protocols for extraction and hydrolysis of DNA were optimized using 54 samples from hepatocellular carcinoma patients with various risk factors, and the 8-oxodG and 2'-deoxyguanosine (dG) levels were measured. The patients enrolled in the study include 23 from The Princess Alexandra Hospital and The Royal Brisbane and Women's Hospitals, Brisbane, Australia, and 31 from South Africa. This study revealed that the 8-oxodG/dG ratios tended to be higher in most non-malignant liver tissues compared to hepatocellular carcinoma tissue (p=0.2887). It also appeared that the ratio was higher in non-malignant liver tissue from Southern African patients (p=0.0479), but there was no difference in the 8-oxodG/dG ratios between non-malignant liver tissues and tumors of Australian hepatocellular carcinoma patients (p=0.7722). Additionally, this study also revealed a trend for a higher 8-oxodG/dG ratio in non-malignant liver tissues compared to tumoural tissues of patients with HBV. Significant differences were not observed in the 8-oxodG/dG ratios between non-cirrhotic and cirrhotic non-malignant liver tissues.

Tobacco smoking and oxidative stress to DNA: a meta-analysis of studies using chromatographic and immunological methods.

Oxidative stress to DNA from smoking was investigated in one randomized smoking cessation study and in 36 cohort studies from excretion of urinary 8-oxo-7-hydrodeoxyguanosine (8-oxodG). Meta-analysis of the 36 cohort studies showed smoking associated with a 15.7% (95% CL 11.0:20.3, p < 0.0001) increased oxidative stress to DNA, in agreement with the reduction of oxidative stress to DNA found in the smoking cessation study. Meta-analysis of the 22 studies that used chromatography methodology on 1709 persons showed a significant 29.3% increase in smokers (95% CL 17.3;41.3), but meta-analysis of 14 studies on 3668 persons using ELISA methodology showed a non-significant effect of 8.7% [95% CL -1.2;18.6]. Tobacco smoke induces oxidative damage to DNA; however, this is not detected with ELISA methodology. Currently, the use of existing ELISA methodology to measure urinary excretion of 8-oxo-7-hydrodeoxyguanosine cannot be recommended.

Evaluation of the oxidative deoxyribonucleic acid damage biomarker 8-hydroxy-2'-deoxyguanosine in the urine of leukemic children by micellar electrokinetic capillary chromatography.

Determining the level of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidative DNA damage biomarker, is vital to the study of clinical pathogenesis and drug toxicity. The principal limitation of capillary electrophoresis (CE) with UV detection is its low sensitivity. To overcome this shortcoming, we developed a micellar electrokinetic capillary chromatography (MEKC) with solid-phase extraction (SPE) for urinary 8-OHdG analysis. The sensitivity of MEKC-UV was improved using a reasonable UV system, injection mode, and SPE. The parameters affecting MEKC and SPE were also optimized. The calibration curve was linear within the range from 1 to 500 µg L(-1). The limits of detection and quantification were 0.27 µg L(-1) and 0.82 µg L(-1), respectively. Interday and intraday precision were both <5.6%. The recovery of 8-OHdG in urine ranged from 94.5% to 103.2%. This method was used to measure urinary 8-OHdG from eight normal children, eight newly diagnosed leukemic children, and eight leukemic children undergoing chemotherapy. The results show that the proposed method can be used to assess oxidative stress in patients and the side effects of chemotherapeutic drugs by measuring urinary 8-OHdG.

Highly sensitive covalently functionalised integrated silicon nanowire biosensor devices for detection of cancer risk biomarker.

In this article we present ultra-sensitive, silicon nanowire (SiNW)-based biosensor devices for the detection of disease biomarkers. An electrochemically induced functionalisation method has been employed to graft antibodies targeted against the prostate cancer risk biomarker 8-hydroxydeoxyguanosine (8-OHdG) to SiNW surfaces. The antibody-functionalised SiNW sensor has been used to detect binding of the 8-OHdG biomarker to the SiNW surface within seconds of exposure. Detection of 8-OHdG concentrations as low as 1 ng/ml (3.5 nM) has been demonstrated. The active device has been bonded to a disposable printed circuit which can be inserted into an electronic readout system as part of an integrated Point of Care (POC) diagnostic. The speed, sensitivity and ease of detection of biomarkers using SiNW sensors render them ideal for eventual POC diagnostics.

Disposable immunosensor using a simple method for oriented antibody immobilization for label-free real-time detection of an oxidative stress biomarker implicated in cancer diseases.

This work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2'-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Non-oriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented methodology favored Ab/Ag affinity and immunodetection of the antigen. The immunosensor design was evaluated by quartz-crystal microbalance with dissipation, atomic force microscopy, electrochemical impedance spectroscopy (EIS) and square-wave voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charge transfer resistance across the electrochemical set-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from glucose, urea and creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted. Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.

Elevated α-methyl-γ-hydroxy-1,N2-propano-2'-deoxyguanosine levels in urinary samples from individuals exposed to urban air pollution.

Acetaldehyde and crotonaldehyde are genotoxic aldehydes present in tobacco smoke and vehicle exhaust. The reaction of these aldehydes with 2'-deoxyguanosine in DNA produces α-methyl-γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine (1,N(2)-propanodGuo). Online HPLC-tandem mass spectrometry was utilized to accurately quantify 1,N(2)-propanodGuo in human urinary samples from 47 residents of São Paulo City (SP) and 35 residents of the rural municipality of São João da Boa Vista (SJBV) in the state of São Paulo. Significantly higher 1,N(2)-propanodGuo levels were found in the samples from SP donors than in samples from SJBV donors. Our results provide the first evidence that elevated levels of 1,N(2)-propanodGuo in urinary samples may be correlated with urban air pollution.

[Chemical constituents from the linseed meal].

Ten compounds were isolated from the 70% ethanol extract of linseed meal (Linum usitatissimum L) through a combination of various chromatographic techniques, including silica gel, macroporous adsorbent resin, Sephadex LH-20, and preparative HPLC. On the basis of spectroscopic data analysis, they were elucidated as 1-methylethyl-2-O-beta-D-glucopyranosyl-(1" --> 6')-beta-D-glucopyanoside (1), linustatin (2), neolinustatin (3), lotaustralin (4), linamarin (5), deoxyguanosine (6), deoxyadenosine (7), (+)-pinoresinol-4'-O-beta-D-glucopyranoside (8), 4-O-beta-D-glucopyranosylvanillyl alcohol (9) and tachioside (10), separately. Among them, compound 1 is a new compound, and compounds 6, 8 and 10 were isolated from the linseed meal for the first time.

Determination of urinary 8-hydroxy-2'-deoxyguanosine by a combination of on-line molecularly imprinted monolithic solid phase microextraction with high performance liquid chromatography-ultraviolet detection.

8-Hydroxy-2'-deoxyguanosine (8-OHdG) is a sensitive biomarker for DNA oxidative damage. However, its determination in human urine is confounded by trace level and complex matrix. In this study, a new configuration of on-line solid phase microextraction coupled to high performance liquid chromatography-ultraviolet detection was established with molecularly imprinted monolithic column as extraction sorbent. The tailor made monolith exhibited high extraction efficiency with the enrichment factor 101.84 for 8-OHdG owing to its special porous structure and inherent selectivity. Under optimal condition, appreciable sensitivity had been achieved for this incorporation with limit of detection 2.04 nmol/L (S/N = 3) and limit of quantification 7.12 nmol/L (S/N = 10), respectively. Precise determination with wide range linearity (0.007-5.00 µmol/L) afforded a practical alternative in urinary 8-OHdG analysis and 107 different subjects had been successfully analyzed. This newly developed method embodied useful prospect for the investigation of DNA oxidative damage with less expense, convenient maintenance and ease of operation.

Gender-related reference intervals of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine determined by liquid chromatography-tandem mass spectrometry in Serbian population.

OBJECTIVES: Although there are many nucleobase modifications, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is one of the dominant form of oxidative modifications of DNA. Urinary 8-oxodG is potentially the best non-invasive biomarker of oxidative stress. Defining reference interval for urinary 8-oxodG is a prerequisite for its clinical use as biomarker. DESIGN AND METHODS: Reference population included 229 healthy Serbian adults (130 males and 99 females). The spot urinary 8-oxodG was determined using high performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS). Urinary creatinine was measured by the kinetic Jaffé method. RESULTS: Analytical performances of the HPLC-MS/MS: CVs within and between-run variations were 5.6% and 2.6%; LOD and LOQ were 1.65 nmol/L and 3.30 nmol/L; mean recovery and relative accuracy were 96% and 97%. Creatinine level was higher in males than in females, but no gender difference in 8-oxodG level was observed. Upon the adjustment of 8-oxodG to creatinine (8-oxodG/creatinine), higher values were obtained in females (1.38 ± 0.65 nmol/mmol) than in males (1.05 ± 0.48 nmol/mmol). Distribution of 8-oxodG/creatinine in spot urine sample was log-normal and gender-related reference intervals (estimated as the 2.5th-97.5th percentiles) were 0.45-2.22 nmol/mmol for males, and 0.54-3.11 nmol/mmol for females. Body mass index (BMI) affects excretion of the 8-oxodG in males, independently of urinary creatinine, while in females it does not. Therefore, BMI might contribute to the gender-related differences of 8-oxodG/creatinine in spot urine samples. CONCLUSIONS: This is the first established gender-related reference intervals of spot urinary 8-oxodG/creatinine. Our results contribute to the full validation of 8-oxodG as biomarker of oxidative stress.

Biochemical investigations into the mutagenic potential of 8-oxo-2'-deoxyguanosine using nucleotide analogues.

8-Oxo-2'-deoxyguanosine (OdG) is an abundant DNA lesion produced during oxidative damage to DNA. It can form relatively stable base pairs with both dC and dA that mimic natural dG:dC and dT:dA base pairs, respectively. Thus, when in the template strand, OdG can direct the insertion of either dCTP or dATP during replication, the latter of which can lead to a dG → T transversion. The potential for OdG to cause mutation is dependent on the preference for dCTP or dATP insertion opposite OdG, as well as the ability to extend past the resulting base pairs. The C2-amine and C8-oxygen could play major roles during these reactions since both would lie outside the Watson-Crick cognate base pairs shape in the major groove when OdG base pairs to dA and dC, respectively, and both have the ability to form strong interactions, like hydrogen bonds. To gain a more generalized understanding of how the C2-amine and C8-oxygen of OdG affect its mutagenic potential, the incorporation opposite and extension past seven analogues of dG/OdG that vary at C2 and/or C8 were characterized for three DNA polymerases, including an exonuclease-deficient version of the replicative polymerase from RB69 (RB69), human polymerase (pol) ß, and polymerase IV from Sulfolobus solfataricus P2 (Dpo4). Based on the results from these studies, as well as those from previous studies with RB69, pol ß, Dpo4, and two A-family polymerases, the influence of the C2-amine and C8-oxygen during each incorporation and extension reaction with each polymerase is discussed. In general, it appears that when the C2-amine and the C8-oxygen are in the minor groove, they allow OdG to retain interactions that are normally present during insertion and extension. However, when the two groups are in the major groove, they each tend to form novel active site interactions, both stabilizing and destabilizing, that are not present during insertion and extension with natural DNA.

Rapid and simple one-step membrane extraction for the determination of 8-hydroxy-2'-deoxyguanosine in human plasma by a combination of on-line solid phase extraction and LC-MS/MS.

A quantitative analytical method using automated on-line solid phase extraction (SPE) and liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) for the determination of 8-OHdG (8-hydroxy-2'-deoxyguanosine) in human plasma was developed and validated. A one-step membrane extraction method for the plasma sample preparation and a C18 SPE column with simple extraction and purification were used for the on-line extraction. A C18 column was employed for LC separation and ESI-MS/MS was utilized for detection. (15)N(5)-8-OHdG ((15)N(5)-8-hydroxy-2'-deoxyguanosine) was used as an internal standard for quantitative determination. The extraction, clean-up and analysis procedures were controlled by a fully automated six-port switch valve as one strategy to reduce the matrix effect and simultaneously improve detection sensitivity. Identification and quantification were based on the following transitions: m/z 284→168 for 8-OHdG and m/z 289→173 for (15)N(5)-8-OHdG. Satisfactory recovery was obtained, and the recovery ranged from 95.1 to 106.1% at trace levels in human plasma and urine, with a CV lower than 5.4%. Values for intraday and interday precision were between 2.3 and 6.8% for plasma and between 2.7 and 4.5% for urine, respectively. Values for the method accuracy of intraday and interday assays ranged from 93.0 and 100.5% for plasma and 110.2 and 119.4% for urine, respectively. The limits of detection (LOD) and LOQ were 0.008 ng/mL and 0.02 ng/mL, respectively.The applicability of this newly developed method was demonstrated by analysis of human plasma samples for an evaluation of the future risk of oxidative stress status in human exposure to nanoparticles and other diseases.

Fused-core silica column ultra-performance liquid chromatography-ion trap tandem mass spectrometry for determination of global DNA methylation status.

Epigenetic modifications, such as DNA methylation, play key roles in transcriptional regulation of gene expression. More recently, global DNA methylation levels have been documented to be altered in several diseases, including cancer, and as the result of exposure to environmental toxicants. Based on the potential use of global DNA methylation status as a biomarker of disease status and exposure to environmental toxicants, we sought to develop a rapid, sensitive, and precise analytical method for the quantitative measurement of global DNA methylation status using ultra-performance liquid chromatography with detection by ion trap tandem mass spectrometry. Using a fused-core silica column, 2'-deoxyguanosine (2dG) and 5-methyl-2'-deoxycytidine (5mdC) were resolved in less than 1 min with detection limits of 0.54 and 1.47 fmol for 5mdC and 2dG, respectively. The accuracy of detection was 95% or higher, and the day-to-day coefficient of variation was found to be 3.8%. The method was validated by quantification of global DNA methylation status following treatment of cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, which reduced DNA methylation from 3.1% in control cells to 1.1% in treated cells. The sensitivity and high throughput of this method rend it suitable for large-scale analysis of epidemiological and clinical DNA samples.

Non-invasive assessment of oxidatively damaged DNA: liquid chromatography-tandem mass spectrometry analysis of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine.

The ability to non-invasively assess DNA oxidation and its repair, has significant utility in large-scale, population-based studies. Such studies could include the assessments of: the efficacy of antioxidant intervention strategies, pathological roles of DNA oxidation in various disease states and population or interindividual differences in antioxidant defence and DNA repair. The most popular method, to non-invasively assess oxidative insult to the genome is by the analysis of urine for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), using chromatographic techniques or immunoassay procedures. The provenance of extracellular 8-oxodG remains a subject for debate. However, previous studies have shown that factors, such as diet and cell death, do not appear to contribute to extracellular 8-oxodG, leaving processes, such as the repair of DNA and/or the 2'-deoxyribonucleotide pool, as the sole source of endogenous 8-oxodG. The method in this chapter describes a non-invasive approach for assessing oxidative stress, via the efficient extraction of urinary 8-oxodG using a validated solid-phase extraction procedure. Subsequent analysis by liquid chromatography-tandem mass spectrometry provides the advantages of sensitivity, internal standardisation, and robust peak identification, and is widely considered to be the "gold standard".

Analysis of 7,8-dihydro-8-oxo-2'-deoxyguanosine in cellular DNA during oxidative stress.

Analysis of cellular 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) as a biomarker of oxidative DNA damage has been fraught with numerous methodological problems. This is primarily due to artifactual oxidation of dGuo that occurs during DNA isolation and hydrolysis. Therefore, it has become necessary to rely on using the comet assay, which is not necessarily specific for 8-oxo-dGuo. A highly specific and sensitive method based on immunoaffinity purification and stable isotope dilution liquid chromatography (LC)-multiple reaction monitoring (MRM)/mass spectrometry (MS) that avoids artifact formation has now been developed. Cellular DNA was isolated using cold DNAzol (a proprietary product that contains guanidine thiocyanate) instead of chaotropic- or phenol-based methodology. Chelex-treated buffers were used to prevent Fenton chemistry-mediated generation of reactive oxygen species (ROS) and artifactual oxidation of DNA bases. Deferoxamine was also added to all buffers in order to complex any residual transition metal ions remaining after Chelex treatment. The LC-MRM/MS method was used to determine that the basal 8-oxo-dGuo level in DNA from human bronchoalveolar H358 cells was 2.2 +/- 0.4 8-oxo-dGuo/10(7) dGuo (mean +/- standard deviation) or 5.5 +/- 1.0 8-oxo-dGuo/10(8) nucleotides. Similar levels were observed in human lung adenocarcinoma A549 cells, mouse hepatoma Hepa-1c1c7 cells, and human HeLa cervical epithelial adenocarcinoma cells. These values are an order of magnitude lower than is typically reported for basal 8-oxo-dGuo levels in DNA as determined by other MS- or chromatography-based assays. H358 cells were treated with increasing concentrations of potassium bromate (KBrO3) as a positive control or with the methylating agent methyl methanesulfonate (MMS) as a negative control. A linear dose-response for 8-oxo-dGuo formation (r(2) = 0.962) was obtained with increasing concentrations of KBrO3 in the range of 0.05 mM to 2.50 mM. In contrast, no 8-oxo-dGuo was observed in H358 cell DNA after treatment with MMS. At low levels of oxidative DNA damage, there was an excellent correlation between a comet assay that measured DNA single strand breaks (SSBs) after treatment with human 8-oxo-guanine glycosylase-1 (hOGG1) when compared with 8-oxo-dGuo in the DNA as measured by the stable isotope dilution LC-MRM/MS method. Availability of the new LC-MRM/MS assay made it possible to show that the benzo[a]pyrene (B[a]P)-derived quinone, B[a]P-7,8-dione, could induce 8-oxo-dGuo formation in H358 cells. This most likely occurred through redox cycling between B[a]P-7,8-dione and B[a]P-7,8-catechol with concomitant generation of DNA damaging ROS. In keeping with this concept, inhibition of catechol-O-methyl transferase (COMT)-mediated detoxification of B[a]P-7,8-catechol with Ro 410961 caused increased 8-oxo-dGuo formation in the H358 cell DNA.